1. Field
The present invention relates to a spectral observation method and a spectral observation system of a specimen containing plural kinds of substances of which exciting wavelengths are different, such as a multiple dyeing sample with plural kinds of fluorescent reagents.
2. Description of the Related Art
A spectral imaging fluorescent laser microscope is effective for an observation of a multiple dyeing sample with plural kinds of fluorescent reagents (refer to Japanese Translation of PCT Publication No. 2004-506191, and so on). This laser microscope irradiates excitation light to the sample, performs a spectral diffraction of fluorescence generated there by a grating (diffraction grating) and so on, and detects intensity of each wavelength component after spectral diffraction by a spectral detector. It is also necessary in this laser microscope to cut excessive excitation light reflected by a surface of the sample, a surface of a lens and so on at any place so as not to prevent the observation, as same as the other fluorescent microscopes.
However, it is necessary to secure a wide wavelength band of the fluorescence in this laser microscope performing the spectral detection, and therefore, a specific channel of the spectral detectors corresponding to an exciting wavelength is excluded from a detection wavelength range of the spectral detector (a masking shield is disposed (fixed) in front of the channel) instead of inserting a barrier filter into an optical path of the fluorescence. When the sample is dyed with two kinds of fluorescent reagents, it is conceivable that each of two channels corresponding to these exciting wavelengths are excluded from the detection wavelength range, and then, these fluorescent reagents are excited simultaneously or sequentially to perform a spectral imaging thereof.
However, there is a possibility in which a part of necessary data may be lost in the spectral imaging as stated above. It is because a necessary wavelength may be excluded from (lost) the detection wavelength range when the exciting wavelength of a certain fluorescent reagent is included in a wavelength range of an emission spectrum of the other fluorescent reagent.